G‐protein‐coupled receptors are integral membrane proteins which constitute the largest family of signal transduction molecules participating in the majority of normal physiological processes. ...G‐protein‐coupled receptors are responsible for the control of enzyme activity, ion channels and vesicle transport, and they respond to a wide variety of stimuli, like signals involved in sensory systems such as vision, taste and olfaction, but also to a diverse set of chemical signals such as lipids, hormones, neurotransmitters, amino acids, nucleotides, peptides and proteins. This family of receptors is being widely studied because of its potential use as pharmacological targets in drug development, and recently also for its potential use in the development of novel biosensors. G‐protein‐coupled receptors are specifically designed to fold and function in a lipid bilayer environment, where these membrane proteins are remarkably stable and achieve their optimal performance. The currently used technology for the purification of G‐protein‐coupled receptors consists in their extraction from the cell membrane and solubilization into detergent micelles. A common drawback of this strategy is that G‐protein‐coupled receptors solubilized in typical detergents show rather poor conformational stability, which may result in relatively rapid inactivation. The poor stability of detergent‐solubilized samples renders many membrane proteins biochemically intractable. This precludes the determination of a high‐resolution structure and imposes severe limitations for the development of applications. Thus, the enhancement of the stability of G‐protein‐coupled receptors is a major issue in order to facilitate structural determination and to unravel their potential in biotechnological applications. This work provides a brief overview of some current advances in the experimental methods for stabilizing G‐protein‐coupled receptors that can also be extended to other types of membrane proteins.
Inorganic salts and novel solubilizing systems enhance the structural stability of membrane proteins opening new avenues for biotechnological applications. Stabilized G‐protein‐coupled receptors can be used for the development of novel biosensors and in biomedical research for clinical diagnostics.
No single molecular mechanism accounts for the effect of mutations in rhodopsin associated with retinitis pigmentosa. Here we report on the specific effect of a Ca super(2) super(+)/recoverin upon ...phosphorylation of the autosomal dominant retinitis pigmentosa R135L rhodopsin mutant. This mutant shows specific features like impaired G-protein signaling but enhanced phosphorylation in the shut-off process. We now report that R135L hyperphosphorylation by rhodopsin kinase is less efficiently inhibited by Ca super(2) super(+)/recoverin than wild-type rhodopsin. This suggests an involvement of Ca super(2) super(+)/recoverin into the molecular pathogenic effect of the mutation in retinitis pigmentosa which is the cause of rod photoreceptor cell degeneration. This new proposed role of Ca super(2) super(+)/recoverin may be one of the specific features of the proposed new Type III class or rhodopsin mutations associated with retinitis pigmentosa.
Detergent-solubilized bovine rhodopsin produces mixed detergent/lipid/protein micelles. The effect of dodecyl maltoside detergent on the thermal stability of dark-state rhodopsin, and upon formation ...of the different intermediates after rhodopsin photobleaching (metarhodopsin II and metarhodopsin III), and upon transducin activation has been studied. No significant effect is observed for the thermal stability of dark-state rhodopsin in the range of detergent concentrations studied, but a decrease in the stability of metarhodopsin II and an increase in metarhodopsin III formation is observed with decreasing detergent concentrations. The transducin activation process is also affected by the presence of detergent indicating that this process is dependent on the lipid micro-environment and membrane fluidity, and this stresses the importance of the native lipid environment in rhodopsin normal function.
Textile industrial scouring processes are currently based on the use of chemical reagents that cause an important environmental impact. For some years, several research groups have focused on ...obtaining enzymes that can carry out the scouring task in an effective way, allowing at the same time a decrease of the industrial costs of water and energy associated with the process. As part of this effort, bioscouring of cotton using enzymes produced in Sclerotium rolfsii has been successfully carried out in the present study. Enzyme production was induced by glucose, glucose-pectin or cellulose, and in all cases the enzyme activity corresponded to polygalacturonase activity. The weight loss after bioscouring was more efficient with enzymes induced by pectin because these have a specific activity for removing pectin content from the cotton fiber. The effectiveness of the enzymatic scouring was equivalent or better than that achieved by the conventional alkaline process.
The study of the structural differences between rhodopsin and its active form (metarhodopsin II) has been carried out by means of deconvolution analysis of infrared spectra. Deconvolution techniques ...allow the direct identification of the spectral changes that have occurred, which results in a significantly different view of the conformational changes occurring after activation of the receptor as compared with previous difference spectroscopy analysis. Thus, a number of changes in the bands assigned to solvent-exposed domains of the receptor are detected, indicating significant decreases in extended (beta) sequences and in reverse turns, and increases in irregular/aperiodic sequences and in helices with a non-alpha geometry, whereas there is no decrease in alpha-helices. In addition to secondary structure conversions, qualitative alterations within a given secondary structure type are detected. These are seen to occur in both reverse turns and helices. The nature of this spectral change is of great importance, since a clear alteration in the helices bundle core is detected. All these changes indicate that the rhodopsin --> metarhodopsin II transition involves not a minor but a major conformational rearrangement, reconciling the infrared data with the energetics of the activation process.
α-Crystallin, one of the major proteins in the vertebrate eye lens, acts as a molecular chaperone, like the small heat-shock proteins, by protecting other proteins from denaturing under stress or ...high temperature conditions. α-Crystallin aggregation is involved in lens opacification, and high Ca
2+ has been associated with cataract formation, suggesting a role for this cation in the pathological process. We have investigated the effect of Ca
2+ on the thermal stability of α-crystallin by UV and Fourier-transform infrared (FTIR) spectroscopies. In both cases, a Ca
2+-induced decrease in the midpoint of the thermal transition is detected. The presence of high Ca
2+ results also in a marked decrease of its chaperone activity in an insulin-aggregation assay. Furthermore, high Ca
2+ concentration decreases Cys reactivity towards a sulfhydryl reagent. The results obtained from the spectroscopic analysis, and confirmed by circular dichroism (CD) measurements, indicate that Ca
2+ decreases both secondary and tertiary–quaternary structure stability of α-crystallin. This process is accompanied by partial unfolding of the protein and a clear decrease in its chaperone activity. It is concluded that Ca
2+ alters the structural stability of α-crystallin, resulting in impaired chaperone function and a lower protective ability towards other lens proteins. Thus, α-crystallin aggregation facilitated by Ca
2+ would play a role in the progressive loss of transparency of the eye lens in the cataractogenic process.
Besides some pharmacological, biochemical and biophysical evidences support the contention that muscarinic acetylcholine receptors can form homo- and heterodimers, the existence of specific M(3) and ...M(5) muscarinic receptors oligomers in living cells is a new concept. Interestingly, this phenomenon might have relevance in lymphocytic cholinergic function since both T- and B-cells naturally express high levels of these two receptor subtypes. Here, by means of co-immunoprecipitation and bioluminescence resonance energy transfer methods we demonstrated that M(3) and M(5) muscarinic receptors could form constitutive homo- and heterodimers in transiently transfected HEK-293T cells. Interestingly, this receptor-receptor interaction was unaltered by carbachol treatment but it was affected by the expression of a peptide corresponding to a portion of the third intracellular loop of the M(5) muscarinic receptor. In addition, the same peptide was able to abrogate the carbachol-induced mitogen-activated protein kinase phosphorylation and the carbachol-enhanced PHA-induced IL-2 production in derived lymphocytic T cells. Overall, these results suggest that the third intracellular loop of the M(5) muscarinic receptor might play a regulatory role in receptor function and heteromerization, thus providing the molecular framework for a potential cholinergic-based therapeutic intervention of the immune system.
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